Barrett's esophagus is a premalignant lesion that increases the risk for esophageal adenocarcinoma. During the last funding period, gene expression analysis revealed that compared to normal esophageal tissue, the AGR2 gene is highly expressed in Barrett's esophagus and esophageal adenocarcinoma. AGR2 is also expressed in a variety of other adenocarcinomas including colon, pancreatic, breast, and prostate cancers. Using RNA interference to repress gene expression in a Barrett's associated adenocarcinoma cell line, SEG-1, we established that AGR2 displays many properties associated with other genes important in cancer. SEG-1 AGR2 knockdown cells show a reduction in colony formation in soft agar and decreased growth as xenografts in nude mice. Tissue culture supernatant containing AGR2 increases cell migration in an in vitro assay. AGR2 also displayed features associated with previously described oncogenes in its ability to transform NIH3T3 cells in in vitro assays such as foci formation and the capacity for anchorage independent growth in soft agar. Studies of AGR2 expression in normal cells revealed that it is expressed in a distribution consistent with areas of cell proliferation in the intestinal crypts throughout the gastrointestinal tract. We have established that intestinal cells of secretory lineage specifically express AGR2. The results suggest that AGR2 may represent a link between gastrointestinal lineage-specific stem cells and cancer. During the next funding period, we propose to elucidate AGR2's mechanism of action. We will define the essential AGR2 protein domains required for its actions, the genomic domains and determinants for its expression, and the genes and proteins whose expression is affected by AGR2. It is likely that AGR2's effects are mediated via activation of signal transduction pathways, the identity of which will also be determined. Studies focused on AGR2 will provide valuable insights into esophageal cancer biology and serves as a promising target for therapeutic intervention. It is also likely that many other adenocarcinomas that express AGR2 will benefit from an understanding of this gene's biology. PUBLIC HEALTH RELEVANCE: Barrett's esophagus is an abnormal lesion commonly found in patients with gastroesophageal reflux, which commonly presents as heartburn. The presence of Barrett's esophagus increases one's risk for esophageal adenocarcinoma by up to 40-fold. Our laboratory recently performed a screen of the entire human genome looking for genes that are active in Barrett's esophagus and adenocarcinoma. We found a gene named AGR2 to be particularly active. Our laboratory has recently found that an active AGR2 gene promotes esophageal cancer cell growth. We also discovered that the AGR2 gene is expressed in the normal gastrointestinal tract in areas often associated with stem cells. These stem cells give rise to all the different cell types in the gastrointestinal tract and are responsible for the normal replacement of aged cells. Current hypotheses propose that deranged regulation of stem cells may give rise to cancer. Additional studies have revealed that cells expressing AGR2 belong to the secretory lineage of the intestine, which is the cell type that develops into adenocarcinomas. The data supports a significant role for AGR2 in the development of esophageal adenocarcinomas. We propose to determine during the next funding period how AGR2 promotes the development of Barrett's esophagus and esophageal adenocarcinoma. We will determine what cellular signals induce AGR2 expression as well as those signaling pathways that are induced by AGR2 itself. Adenocarcinomas are glandular cancers that are also common in the colon, pancreas, breast and prostate. Because the AGR2 gene is active in all these cancers, studies focused on this gene will increase our understanding of the mechanisms underlying many cancers and provide an opportunity for developing new therapeutic strategies.